Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes.

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

A roadmap for therapeutic discovery in pulmonary hypertension associated with left heart failure. A scientific statement of the Heart Failure Association (HFA) of the ESC and the ESC Working Group on Pulmonary Circulation & Right Ventricular Function.

Pulmonary hypertension (PH) associated with left heart failure (LHF) (PH-LHF) is one of the most common causes of PH. It directly contributes to symptoms and reduced functional capacity and negatively affects right heart function, ultimately leading to a poor prognosis. There are no specific treatments for PH-LHF, despite the high number of drugs tested so far. This scientific document addresses the main knowledge gaps in PH-LHF with emphasis on pathophysiology and clinical trials. Key identified issues include better understanding of the role of pulmonary venous versus arteriolar remodelling, multidimensional phenotyping to recognize patient subgroups positioned to respond to different therapies, and conduct of rigorous pre-clinical studies combining small and large animal models. Advancements in these areas are expected to better inform the design of clinical trials and extend treatment options beyond those effective in pulmonary arterial hypertension. Enrichment strategies, endpoint assessments, and thorough haemodynamic studies, both at rest and during exercise, are proposed to play primary roles to optimize early-stage development of candidate therapies for PH-LHF.

Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival.

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation.

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

The role of fibrosis in cardiomyopathies: An opportunity to develop novel biomarkers of disease activity.

Cardiomyopathies encompass a spectrum of heart disorders with diverse causes and presentations. Fibrosis stands out as a shared hallmark among various cardiomyopathies, reflecting a common thread in their pathogenesis. This prevalent fibrotic response is intricately linked to the consequences of dysregulated extracellular matrix (ECM) remodeling, emphasizing its significance in the development and progression the disease. This review explores the ECM involvement in various cardiomyopathies and its impact on myocardial stiffness and fibrosis. Additionally, we discuss the potential of ECM fragments as early diagnosis, prognosis, and risk stratification. Biomarkers deriving from turnover of collagens and other ECM proteins hold promise in clinical applications. We outline current clinical management, future directions, and the potential for personalized ECM-targeted therapies with specific focus on microRNAs. In summary, this review examines the role of the fibrosis in cardiomyopathies, highlighting the potential of ECM-derived biomarkers in improving disease management with implications for precision medicine.

Circulating miR-let7a levels predict future diagnosis of chronic thromboembolic pulmonary hypertension.

Distinct patterns of circulating microRNAs (miRNAs) were found to be involved in misguided thrombus resolution. Thus, we aimed to investigate dysregulated miRNA signatures during the acute phase of pulmonary embolism (PE) and test their diagnostic and predictive value for future diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH). Microarray screening and subsequent validation in a large patient cohort (n = 177) identified three dysregulated miRNAs as potential biomarkers: circulating miR-29a and miR-720 were significantly upregulated and miR-let7a was significantly downregulated in plasma of patients with PE. In a second validation study equal expression patterns for miR-29a and miR-let7a regarding an acute event of recurrent venous thromboembolism (VTE) or deaths were found. MiR-let7a concentrations significantly correlated with echocardiographic and laboratory parameters indicating right ventricular (RV) dysfunction. Additionally, circulating miR-let7a levels were associated with diagnosis of CTEPH during follow-up. Regarding CTEPH diagnosis, ROC analysis illustrated an AUC of 0.767 (95% CI 0.54-0.99) for miR-let7a. Using logistic regression analysis, a calculated patient-cohort optimized miR-let7a cut-off value derived from ROC analysis of ≥ 11.92 was associated with a 12.8-fold increased risk for CTEPH. Therefore, miR-let7a might serve as a novel biomarker to identify patients with haemodynamic impairment and as a novel predictor for patients at risk for CTEPH.

A pilot study for risk stratification of ventricular tachyarrhythmia in hypertrophic cardiomyopathy with routine echocardiography parameters.

Ventricular tachyarrhythmia (VTA) are frequent arrhythmias in patients with hypertrophic cardiomyopathy (HCM). Representing a major risk factor for sudden cardiac death, Holter ECG at first clinical presentation appears insufficient. This study aims to investigate the ability of routinely obtained parameters associated with myocardial remodeling in stratifying for VTA in HCM. In this monocentric analysis, patients with HCM underwent 12-channel electrocardiography and echocardiography, including tissue doppler imaging. The study's primary endpoint was the documentation of non-sustained and sustained ventricular tachycardia-summarized as ventricular tachyarrhythmias (VTA) on Holter ECG or active devices. The occurrence of VTA was exploratory. Based on our collective, we developed a risk model regarding VTA. Of 140 HCM patients, 38 (27.1%) had an episode of VTA. Patients with VTA were likelier to have a history of atrial fibrillation (p < 0.001), a thicker interventricular septum (p < 0.001) and lower peak systolic mitral annular velocity (p < 0.001). The parameters were independently associated with endpoint in univariate and multivariate logistic regression. We created a logistic equation and calculated a cut-off value. The resulting ROC curve revealed a discriminative ability with AUC of 0.80 (sensitivity, 63%; specificity, 88%). Our risk model including these widely available parameters is able to distinguish low and high-risk of VTA in patients with HCM.

Encapsulating In Vitro Transcribed circRNA into Lipid Nanoparticles Via Microfluidic Mixing.

This chapter serves as a guide for researchers embarking on circular RNA-based translational studies. It provides a foundation for the successful encapsulation of circular RNA into lipid nanoparticles (LNPs) and facilitates progress in this emerging field. Crucial scientific methods and techniques involved in the formulation process, particle characterization, and downstream processing of circ-LNPs are covered. The production of in vitro transcribed circular RNA-containing LNPs based on a commercially available lipid mix is provided, in addition to the fundamentals for successful encapsulation based on lipid mixes composed of single components. Furthermore, the transfection and validation protocols for the identification of a functional and potentially therapeutic circRNA candidate for initial in vitro verification, before subsequent LNP studies, are explained.

Integration of implantable device therapy in patients with heart failure. A clinical consensus statement from the Heart Failure Association (HFA) and European Heart Rhythm Association (EHRA) of the European Society of Cardiology (ESC).

Implantable devices form an integral part of the management of patients with heart failure (HF) and provide adjunctive therapies in addition to cornerstone drug treatment. Although the number of these devices is growing, only few are supported by robust evidence. Current devices aim to improve haemodynamics, improve reverse remodelling, or provide electrical therapy. A number of these devices have guideline recommendations and some have been shown to improve outcomes such as cardiac resynchronization therapy, implantable cardioverter-defibrillators and long-term mechanical support. For others, more evidence is still needed before large-scale implementation can be strongly advised. Of note, devices and drugs can work synergistically in HF as improved disease control with devices can allow for further optimization of drug therapy. Therefore, some devices might already be considered early in the disease trajectory of HF patients, while others might only be reserved for advanced HF. As such, device therapy should be integrated into HF care programmes. Unfortunately, implementation of devices, including those with the greatest evidence, in clinical care pathways is still suboptimal. This clinical consensus document of the Heart Failure Association (HFA) and European Heart Rhythm Association (EHRA) of the European Society of Cardiology (ESC) describes the physiological rationale behind device-provided therapy and also device-guided management, offers an overview of current implantable device options recommended by the guidelines and proposes a new integrated model of device therapy as a part of HF care.

Dextrans, Pullulan and Lentinan, New Scaffold Materials for Use as Hydrogels in Tissue Engineering.

The development of hydrogels based on dextrans, pullulan and lentinan to be used in biomedical applications including tissue engineering is reported. Despite the fact that selected polysaccharides such as hyaluronic acid are well established, little is known, how these polysaccharides can be chemically modified to create hydrogels under controlled conditions. In this study we present a small library of chemically modified polysaccharides which are used for a divergent approach to achieve biomedical relevant hydrogels. In this case the crosslinking is based on thio ether formation between thiol modified donor and vinylsulfone or maleimide modified acceptor components. Successful synthesis of the linker systems and coupling at the polysaccharides, hydrogel formation takes place under physiological conditions. We extended the study by coupling small molecules like adhesion factors for increasing cell compatibility as well as a dye for further studies. The different hydrogels were studied to their rheological properties, water uptake, their permeability, biodegrability and their cytotoxicity.

Macrophage-based therapeutic approaches for cardiovascular diseases.

Despite the advances in treatment options, cardiovascular disease (CVDs) remains the leading cause of death over the world. Chronic inflammatory response and irreversible fibrosis are the main underlying pathophysiological causes of progression of CVDs. In recent decades, cardiac macrophages have been recognized as main regulatory players in the development of these complex pathophysiological conditions. Numerous approaches aimed at macrophages have been devised, leading to novel prospects for therapeutic interventions. Our review covers the advancements in macrophage-centric treatment plans for various pathologic conditions and examines the potential consequences and obstacles of employing macrophage-targeted techniques in cardiac diseases.

LIPCAR levels in plasma-derived extracellular vesicles is associated with left ventricle remodeling post-myocardial infarction.

BACKGROUND: Long Intergenic noncoding RNA predicting CARdiac remodeling (LIPCAR) is a long noncoding RNA identified in plasma of patients after myocardial infarction (MI) to be associated with left ventricle remodeling (LVR). LIPCAR was also shown to be a predictor of early death in heart failure (HF) patients. However, no information regarding the expression of LIPCAR and its function in heart as well as the mechanisms involved in its transport to the circulation is known. The aims of this study are (1) to characterize the transporter of LIPCAR from heart to circulation; (2) to determine whether LIPCAR levels in plasma isolated-extracellular vesicles (EVs) reflect the alteration of its expression in total plasma and could be used as biomarkers of LVR post-MI. METHODS: Since expression of LIPCAR is restricted to human species and the limitation of availability of cardiac biopsy samples, serum-free conditioned culture media from HeLa cells were first used to characterize the extracellular transporter of LIPCAR before validation in EVs isolated from human cardiac biopsies (non-failing and ischemic HF patients) and plasma samples (patients who develop or not LVR post-MI). Differential centrifugation at 20,000g and 100,000g were performed to isolate the large (lEVs) and small EVs (sEVs), respectively. Western blot and nanoparticle tracking (NTA) analysis were used to characterize the isolated EVs. qRT-PCR analysis was used to quantify LIPCAR in all samples. RESULTS: We showed that LIPCAR is present in both lEVs and sEVs isolated from all samples. The levels of LIPCAR are higher in lEVs compared to sEVs isolated from HeLa conditioned culture media and cardiac biopsies. No difference of LIPCAR expression was observed in tissue or EVs isolated from cardiac biopsies obtained from ischemic HF patients compared to non-failing patients. Interestingly, LIPCAR levels were increased in lEVs and sEVs isolated from MI patients who develop LVR compared to patients who did not develop LVR. CONCLUSION: Our data showed that large EVs are the main extracellular vesicle transporter of LIPCAR from heart into the circulation independently of the status, non-failing or HF, in patients. The levels of LIPCAR in EVs isolated from plasma could be used as biomarkers of LVR in post-MI patients.

Efficacy and safety of CDR132L in patients with reduced left ventricular ejection fraction after myocardial infarction: Rationale and design of the HF-REVERT trial.

AIM: Inhibition of microRNA (miR)-132 effectively prevents and reverses adverse cardiac remodelling, making it an attractive heart failure (HF) target. CDR132L, a synthetic antisense oligonucleotide selectively blocking pathologically elevated miR-132, demonstrated beneficial effects on left ventricular (LV) structure and function in relevant preclinical models, and was safe and well tolerated in a Phase 1b study in stable chronic HF patients. Patients with acute myocardial infarction (MI) and subsequent LV dysfunction and remodelling have limited therapeutic options, and may profit from early CDR132L treatment. METHODS: The HF-REVERT (Phase 2, multicenter, randomized, parallel, 3-arm, placebo-controlled Study to Assess Efficacy and Safety of CDR132L in Patients with Reduced Left Ventricular Ejection Fraction after Myocardial Infarction) evaluates the efficacy and safety of CDR132L in HF patients post-acute MI (n = 280), comparing the effect of 5 and 10 mg/kg CDR132L, administered as three single intravenous doses 28 days apart, in addition to standard of care. Key inclusion criteria are the diagnosis of acute MI, the development of systolic dysfunction (LV ejection fraction ≤45%) and elevated N-terminal pro-B-type natriuretic peptide. The study consists of a 6-month double-blinded treatment period with the primary endpoint LV end-systolic volume index and relevant secondary endpoints, followed by a 6-month open-label observation period. CONCLUSION: The HF-REVERT trial may underpin the concept of miR-132 inhibition to prevent or reverse cardiac remodelling in post-MI HF. The results will inform the design of subsequent outcome trials to test CDR132L in HF.

Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome.

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA.

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 is known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains largely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the heart cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified Msi2 isoforms 1, 4, and 5, and two novel putative isoforms labeled as Msi2 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 exhibited a significant increase in a pressure-overload model of cardiac hypertrophy. We selected isoforms 4 and 7 to validate the hypertrophic effects due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, seahorse, and transmembrane potential measurement assays, increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh and Smyd1 inhibited Msi2-induced cardiac malfunction and mitochondrial dysfunction. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

Acute heart failure: mechanisms and pre-clinical models-a Scientific Statement of the ESC Working Group on Myocardial Function.

While chronic heart failure (CHF) treatment has considerably improved patient prognosis and survival, the therapeutic management of acute heart failure (AHF) has remained virtually unchanged in the last decades. This is partly due to the scarcity of pre-clinical models for the pathophysiological assessment and, consequently, the limited knowledge of molecular mechanisms involved in the different AHF phenotypes. This scientific statement outlines the different trajectories from acute to CHF originating from the interaction between aetiology, genetic and environmental factors, and comorbidities. Furthermore, we discuss the potential molecular targets capable of unveiling new therapeutic perspectives to improve the outcome of the acute phase and counteracting the evolution towards CHF.

Incidence, risk assessment and prevention of sudden cardiac death in cardiomyopathies.

Cardiomyopathies are a significant contributor to cardiovascular morbidity and mortality, mainly due to the development of heart failure and increased risk of sudden cardiac death (SCD). Despite improvement in survival with contemporary treatment, SCD remains an important cause of mortality in cardiomyopathies. It occurs at a rate ranging between 0.15% and 0.7% per year (depending on the cardiomyopathy), which significantly surpasses SCD incidence in the age- and sex-matched general population. The risk of SCD is affected by multiple factors including the aetiology, genetic basis, age, sex, physical exertion, the extent of myocardial disease severity, conduction system abnormalities, and electrical instability, as measured by various metrics. Over the past decades, the knowledge on the mechanisms and risk factors for SCD has substantially improved, allowing for a better-informed risk stratification. However, unresolved issues still challenge the guidance of SCD prevention in patients with cardiomyopathies. In this review, we aim to provide an in-depth discussion of the contemporary concepts pertinent to understanding the burden, risk assessment and prevention of SCD in cardiomyopathies (dilated, non-dilated left ventricular, hypertrophic, arrhythmogenic right ventricular, and restrictive). The review first focuses on SCD incidence in cardiomyopathies and then summarizes established and emerging risk factors for life-threatening arrhythmias/SCD. Finally, it discusses validated approaches to the risk assessment and evidence-based measures for SCD prevention in cardiomyopathies, pointing to the gaps in evidence and areas of uncertainties that merit future clarification.

Circulating miR-133a-3p defines a low-risk subphenotype in patients with heart failure and central sleep apnea: a decision tree machine learning approach.

BACKGROUND: Patients with heart failure with reduced ejection fraction (HFrEF) and central sleep apnea (CSA) are at a very high risk of fatal outcomes. OBJECTIVE: To test whether the circulating miRNome provides additional information for risk stratification on top of clinical predictors in patients with HFrEF and CSA. METHODS: The study included patients with HFrEF and CSA from the SERVE-HF trial. A three-step protocol was applied: microRNA (miRNA) screening (n = 20), technical validation (n = 60), and biological validation (n = 587). The primary outcome was either death from any cause, lifesaving cardiovascular intervention, or unplanned hospitalization for worsening of heart failure, whatever occurred first. MiRNA quantification was performed in plasma samples using miRNA sequencing and RT-qPCR. RESULTS: Circulating miR-133a-3p levels were inversely associated with the primary study outcome. Nonetheless, miR-133a-3p did not improve a previously established clinical prognostic model in terms of discrimination or reclassification. A customized regression tree model constructed using the Classification and Regression Tree (CART) algorithm identified eight patient subphenotypes with specific risk patterns based on clinical and molecular characteristics. MiR-133a-3p entered the regression tree defining the group at the lowest risk; patients with log(NT-proBNP) ≤ 6 pg/mL (miR-133a-3p levels above 1.5 arbitrary units). The overall predictive capacity of suffering the event was highly stable over the follow-up (from 0.735 to 0.767). CONCLUSIONS: The combination of clinical information, circulating miRNAs, and decision tree learning allows the identification of specific risk subphenotypes in patients with HFrEF and CSA.

Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the ß-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively ß-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than ß-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/ß-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted ß-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.